Transcriptome Analysis Reveals the Importance of the Immune System During Early Pregnancy in Sheep

bioRxiv preprint doi: https://doi.org/10.1101/673558; this version posted November 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Transcriptome analysis reveals the importance of the immune system during early pregnancy in sheep

1 Kisun Pokharel1, Jaana Peippo1, Melak Weldenegodguad1,2, Mervi Honkatukia3, Meng-Hua 2 Li4*, Juha Kantanen1*

3 1Natural Resources Institute of Finland (Luke), Jokioinen, Finland 4 2Department of Environmental and Biological Sciences, Faculty of Science and Forestry, University 5 of Eastern Finland, Kuopio, Finland 6 3NordGen – The Nordic Genetic Resources Center, Ås, Norway 7 4CAS Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese 8 Academy of Sciences (CAS), Beijing, China

9 * Correspondence: 10 Prof. Meng-Hua Li 11 [email protected] 12 Prof. Juha Kantanen 13 [email protected]

14 Keywords: Finnsheep, Texel, progesterone, immunoglobulins, Siglec, endogenous retrovirus, 15 endometrium, corpus luteum, prolificacy, preimplantation

16 Running title: Sheep preimplantation transcriptome

17 1 Abstract

18 The majority of pregnancy loss in ruminants occurs during the preimplantation stage, which is thus 19 the most critical period determining reproductive success. While ovulation rate is the major 20 determinant of litter size in sheep, interactions among the conceptus, corpus luteum and endometrium 21 are essential for pregnancy success. To evaluate the role of reproductive tract function in sheep 22 fertility, we performed a comparative transcriptome study by sequencing total RNA (mRNA and 23 miRNA) from corpus luteum (CL) and endometrium tissues collected during the preimplantation 24 stage of pregnancy in Finnsheep, Texel and F1 crosses. A total of 21,287 genes and 599 miRNAs 25 were expressed in our dataset. Ten out of the top 25 most highly expressed genes were shared across 26 tissues, indicating the complementary functions of the CL and endometrium. Moreover, highly 27 expressed autosomal genes in the endometrium and CL were associated with biological processes 28 such as progesterone formation (STAR and HSD3B1) in the CL and facilitation of maternal 29 recognition of pregnancy, trophoblast elongation and implantation (LGALS15, CST3, CST6, and 30 EEF1A1) in the endometrium. In the CL, a group of sialic acid-binding immunoglobulin (Ig)-like 31 lectins (Siglecs), solute carriers (SLC13A5, SLC15A2, SLC44A5) and chemokines (CCL5, CXCL13, 32 CXCL9) were upregulated in Finnsheep, while several multidrug resistance-associated proteins 33 (MRPs) were upregulated in Texel ewes. We also identified a novel ERV gene located in a reduced 34 FecL locus that is associated with sheep prolificacy and is upregulated in prolific Finnsheep. 35 Moreover, we report, for the first time in any species, several genes that are active in the CL during 36 early pregnancy (including SIGLEC13, SIGLEC14, SIGLEC6, MRP4, and CA5A). Importantly, 37 functional analysis of differentially expressed genes suggested that Finnsheep have a better immune bioRxiv preprint doi: https://doi.org/10.1101/673558; this version posted November 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International Sheeplicense. preimplantation transcriptome

38 system than Texel and that high prolificacy in Finnsheep might be governed by immune system 39 regulation. Taken together, the findings of this study provide new insights into the interplay between 40 the CL and the endometrium in gene expression dynamics during early pregnancy. The data and 41 results will serve as a basis for studying this highly critical period of pregnancy, which has wide 42 significance in mammalian fertility and reproduction.

43 2 Introduction

44 Litter size, a key determinant for the profitability of sheep production systems, is highly dependent 45 on ovulation rate and embryo development in the uterus. Earlier studies have shown that the trait of 46 high prolificacy can result due to the action of either a single gene with a major effect, as in the 47 Chinese Hu, Boorola Merino, Lacaune and small-tailed Han breeds (Mulsant et al., 2001; Souza et 48 al., 2001; Davis et al., 2002, 2006; Chu et al., 2007; Drouilhet et al., 2013), or different sets of genes, 49 as in the Finnsheep and Romanov breeds (Ricordeau et al., 1990; Xu et al., 2018). The Finnsheep or 50 Finnish landrace, one of the most highly prolific breeds, has been exported to more than 40 countries 51 to improve local breeds, although the heritability of ovulation rate is low (Hanrahan and Quirke, 52 1984). In recent years, a FecGF (V371M) mutation in gene GDF9 has been identified to be strongly 53 associated with litter size in Finnsheep and breeds such as the Norwegian White Sheep, Cambridge 54 and Belclare breeds, which were developed using Finnsheep (Hanrahan et al., 2004; Våge et al., 55 2013; Mullen and Hanrahan, 2014; Pokharel et al., 2018).

56 The success of pregnancy establishment in sheep and other domestic ruminants is determined at the 57 preimplantation stage and involves coordination among pregnancy recognition, implantation and 58 placentation, in which the corpus luteum (CL) and endometrium play vital roles (Geisert et al., 1992; 59 Spencer et al., 2004b, 2007). The preimplantation stage of pregnancy is the most critical period in 60 determining the litter size because of the high embryo mortality during this period. It has been shown 61 that most embryonic deaths occur before day 18 of pregnancy in sheep (Quinlivan et al., 1966; Bolet, 62 1986; Rickard et al., 2017). However, due to the biological complexity of the process and to technical 63 difficulties, embryo implantation is still not well understood.

64 The CL is an endocrine structure whose main function is to synthesize and secrete the hormone 65 progesterone. Progesterone production is essential for the establishment of pregnancy. However, if 66 pregnancy is not established, the CL will regress as a result of luteolysis, and a new cycle will begin. 67 The endometrium is the site of blastocyst implantation, but its function is not limited to implantation. 68 The outer lining of the endometrium secretes histotroph, a complex mixture of enzymes, growth 69 factors, hormones, transport proteins and other substances that are key to conceptus survival and 70 implantation, pregnancy recognition signal production and placentation (Spencer and Bazer, 2004; 71 Forde et al., 2013). In addition, the endometrium also plays an important role in regulating the 72 estrous cycle (Spencer et al., 2008).

73 The whole-transcriptome profiling approach enables a deeper understanding of the functions of both 74 the CL and endometrium, which may allow the identification of genes and markers that are 75 differentially expressed, for example, between breeds showing different litter size phenotypes. 76 Although most of the studies associated with early pregnancy have been performed in sheep (Spencer 77 et al., 2004b, 2007; Mamo et al., 2012; Bazer, 2013; Raheem, 2017), only a few studies have applied 78 transcriptomic approaches to the endometrium and CL. A microarray-based transcriptomic study 79 conducted by Gray et al. (2006) identified a number of endometrial genes regulated by progesterone 80 (from the CL) and interferon tau (IFNT; from the conceptus) in pregnant vs uterine gland knockout 81 (UGKO) ewes. In a more comprehensive study conducted by Brooks et al. (2016), transcriptome

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82 analysis of uterine epithelial cells during the peri-implantation period of pregnancy identified various 83 regulatory pathways and biological processes in sheep. Moore et al. (2016) combined gene 84 expression data with genome-wide association studies (GWASs) to understand the roles of CL and 85 endometrium transcriptomes in dairy cattle fertility. A study by (Kfir et al., 2018) identified 86 differentially expressed genes (DEGs) between Day 4 and Day 11 in the CL in cattle. Recently, a 87 study on endometrial gene expression differences between Finnsheep and European mouflon 88 identified several genes associated with reproductive processes (Yang et al., 2018). Though these 89 studies have certainly enhanced our understanding of the roles of the CL and endometrium during 90 early pregnancy and in ruminant fertility in general, none of the studies have conducted specific 91 comparisons between breeds with different reproductive potential. Thus, in this study, a comparison 92 of transcriptome profiles between two breeds was conducted to provide insight into the similarities in 93 developmental events in early pregnancy between the breeds. Using F1 crosses we were able to better 94 understand the heritability of the genetic markers. Here, the main goal of this study was to build a 95 global picture of transcriptional complexity in two tissues (Cl and endometrium) and examine 96 differences in developmental profiles during early pregnancy in sheep breeds showing contrasting 97 fertility phenotypes. Thus, this study has relevance to sheep breeding towards achieving improved 98 reproductive capacity.

99 3 Materials and Methods

100 3.1 Experimental design 101 All procedures for the experiment and sheep sampling were approved by the Southern Finland 102 Animal Experiment Committee (approval no. ESAVI/5027/04.10.03/2012). The animals were kept at 103 Pusa Farm in Urjala, located in the province of Western Finland, during the experimental period. A 104 total of 31 ewes representing three breed groups (Finnsheep (n=11), Texel (n=11) and F1 crosses 105 (n=9) were included in the main experiment (please note that only 18 of the 31 ewes have been 106 included in this study). Analyses were conducted for two different time points during the 107 establishment of pregnancy: the follicular growth phase (Pokharel et al., 2018) and early pregnancy 108 prior to implantation (current study). After ovary removal (Pokharel et al., 2018), the ewes were 109 mated using two Finnsheep rams, and the pregnant ewes were slaughtered during the preimplantation 110 phase of the pregnancy when the embryos were estimated to be one to three weeks old. At the 111 slaughterhouse, a set of tissue samples (the pituitary gland, a CL, oviductal and uterine epithelial 112 cells, and preimplantation embryos) were collected and stored in RNAlater reagent (Ambion/Qiagen, 113 Valencia, CA, USA) following the manufacturer’s instructions. Of the collected tissue samples, CL 114 and endometrium tissues were subjected to current study. Endometrial samples were collected from 115 the uterine horns with a cytobrush, which was rinsed in a tube containing RNAprotect Cell Reagent 116 (Qiagen, Valencia, CA, USA). One of the CLs was dissected from each ovary. For the present study, 117 and particularly for the RNA-Seq of the endometrium and CL, six ewes each from the Finnsheep, 118 Texel and F1 cross groups were included. Therefore, out of 31 ewes that were originally included in 119 the main experiment, only 18 have been considered here. The experimental design have been 120 described in more detail in an earlier study (Pokharel et al., 2018).

121 3.2 Library preparation and sequencing 122 Both mRNA and miRNA were extracted from the tissues using an RNeasy Plus Mini Kit (Qiagen, 123 Valencia, CA, USA) following the manufacturer’s protocol. The details on RNA extraction have 124 been described previously (Hu et al., 2015; Pokharel et al., 2018). RNA quality (RNA concentration 125 and RNA integrity number) was measured using a Bioanalyzer 2100 (Agilent Technologies, 126 Waldbronn, Germany) before sending the samples to the Finnish Microarray and Sequencing Center,

127 Turku, Finland, where library preparation and sequencing were performed. RNA libraries were 128 prepared according to the Illumina TruSeq® Stranded mRNA Sample Preparation Guide (part # 129 15031047) which included poly-A selection step. Unique Illumina TruSeq indexing adapters were 130 ligated to each sample during an adapter ligation step to enable pooling of multiple samples into one 131 flow cell lane. The quality and concentrations of the libraries were assessed with an Agilent 132 Bioanalyzer 2100 and by Qubit® Fluorometric Quantitation, Life Technologies, respectively. All 133 samples were normalized and pooled for automated cluster preparation at an Illumina cBot station. 134 High-quality libraries of mRNA and miRNA were sequenced with an Illumina HiSeq 2000 135 instrument using paired-end (2x100 bp) and single-end (1x50) sequencing strategies, respectively.

136 3.3 Data preprocessing and mapping for mRNA 137 The raw reads were assessed for errors and the presence of adapters using FastQC v0.11.6 (Simon 138 Andrews). As we noticed the presence of adapters, Trim Galore v0.5.0 (Felix Krueger; Martin, 2011) 139 was used to remove the adapters and low-quality reads and bases. The transcripts were quantified 140 under the quasi-mapping-based mode in Salmon v0.11.2 (Patro et al., 2017). We extracted the 141 FASTA sequences (oar31_87.fa) of the sheep transcriptome (oar31_87.gtf) using the gffread utility 142 (Trapnell et al., 2010) and built the transcriptome index. The resulting index was used for transcript 143 quantification (also known as pseudo alignment) of the RNA-Seq reads.

144 3.4 Data preprocessing and analysis for miRNA 145 The raw sequence data were initially screened to obtain an overview of the data quality, including the 146 presence or absence of adapters, using FastQC v0.11.6 (Simon Andrews). Next, the Illumina adapters 147 and low-quality bases were removed using Trim Galore v0.5.0 (Felix Krueger; Martin, 2011). In 148 addition, reads that were too short (having fewer than 18 bases) after trimming were also discarded. 149 To reduce downstream computational time, high-quality reads were collapsed using Seqcluster 150 v1.2.4a7 (Pantano et al., 2011). The FASTQ output from Seqcluster was first converted into a 151 FASTA file. The FASTA header was reformatted by including a sample-specific three letter code, 152 which is also a requirement for miRDeep2 analysis. For instance, “>A01_1_x446 A01” represents 153 sample C1033, whose first read was repeated 446 times.

154 The collapsed reads were mapped against the ovine reference genome (oar v3.1) using Bowtie 155 (Langmead et al., 2009). The Bowtie parameters were adjusted so that (1.) the resulting alignments 156 had no more than 1 mismatch (-v 1); (2.) the alignments for a given read were suppressed if more 157 than 8 alignments existed for it (-m 8); and (3.) the best-aligned read was reported (--strata, --best). 158 The alignment outputs (in SAM format) were coordinate-sorted and converted to BAM files. The 159 sorted BAM files were converted to the miRDeep2 ARF format using the “bwa_sam_converter.pl” 160 script.

161 miRDeep2 v2.0.0.5 (Friedländer et al., 2012) was used to identify known ovine miRNAs and to 162 predict conserved (known in other species) and novel ovine miRNAs. Before running the miRDeep2 163 pipeline, we merged both the collapsed FASTA files and the mapped ARF files. Furthermore, hairpin 164 and mature sequences of all species were extracted from miRBase v22 (Kozomara and Griffiths- 165 Jones, 2011, 2014). The extracted sequences were grouped into mature ovine sequences, ovine 166 hairpin sequences, and mature sequences for all species except sheep. The results from miRDeep2 167 were further processed to compile a list of all known and novel miRNAs. For novel and conserved 168 miRNAs, we designated provisional IDs that included the genomic coordinates of the putative mature 169 and star sequences.

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170 3.5 Differential expression of RNA 171 For mRNA-Seq data, the gene expression estimates from Salmon were used to identify DEGs. The 172 Salmon-based transcript counts were summarized to gene level estimates using tximport (Soneson et 173 al., 2016).DESeq2 (Love et al., 2013) was used to compare gene expression differences. We started 174 by considering both tissues, but after observing the high variation between the endometrial samples, 175 we decided to analyze the two tissues separately. Furthermore, PCA plot (Fig. 2A) illustrated a high 176 variation in gene expression estimates between the endometrial samples while tight clustering of the 177 CL samples. Therefore, owing to sampling bias (explanation in results and discussion section), we 178 did not proceed with differential gene expression analysis on endometrial samples. All replicates 179 were collapsed before running DESeq. We set the filtering criteria for significant DEGs to an 180 adjusted p-value of 0.1 (padj <1) and an absolute log2(fold change) of greater than 1 181 (abs(log2Foldchange)>1). All the significant DEGs were annotated with Bioconductor biomaRt 182 (Durinck et al., 2005) to retrieve additional information (gene name, gene description, Entrez ID, 183 human ortholog and chromosome number).

184 From the list of miRNAs discovered with miRDeep2, those with a minimum count of 10 reads across 185 all samples were considered for expression analysis. We used DESeq2 for expression analysis, in 186 which the technical replicates of three samples (C107, C4271 and C312) were collapsed prior to 187 running the DESeq command. Because of the sampling bias in endometrium samples, we did not 188 conduct breed wise differential expression analysis for endometrium samples. The differentially 189 expressed miRNAs with adjusted p-values less than 0.1 were regarded as significant.

190 3.6 Gene ontology and pathway analysis 191 The ClueGO v2.5.3 (Bindea et al., 2009) plugin in Cytoscape v3.7.0 (Shannon et al., 2003) was 192 employed for gene functional analysis. Prior to performing the analyses, we downloaded the latest 193 versions of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology 194 (GO) terms. In addition, we retrieved Entrez gene IDs for all expressed genes in our dataset using the 195 biomaRt Bioconductor package. The enrichment analysis was based on a two-sided hypergeometric 196 test with the Bonferroni step-down correction method. We used a custom reference set that included 197 a list of all the expressed genes in our dataset. We also modified the default GO and pathway 198 selection criteria in such a way that a minimum of three genes and three percent of genes from a 199 given GO or KEGG pathway should be present in the query list. Furthermore, GO terms with a 200 minimum level of three and a maximum level of 5 were retained.

201 3.7 Manual annotation of select genes 202 When we noticed that many genes lacked gene annotations, we manually annotated those among the 203 top 25 most highly expressed genes in each tissue and the significant DEGs. First, we extracted the 204 coding sequence of each novel gene using Ensembl BioMart. All genes that had coding sequences 205 were BLASTed against the nonredundant (NR) nucleotide database. For the BLAST-based 206 annotation, we chose the hit with the highest coverage and the highest percentage of sequence 207 identity to the query sequence. Gene IDs that lacked coding sequences (CDSs) were queried back to 208 the Ensembl database to retrieve existing information. Throughout the paper (including in the 209 supplementary data files), the genes that were annotated based on the BLAST results have been 210 marked with an asterisk (*), while those that were annotated based on information available in 211 Ensembl are marked with a hash (#).

212 4 Results and Discussion

213 4.1 Phenotypic observations 214 After removal of the remaining ovary, we counted the number of CLs visually in each animal. With 215 an average of 4.09, Finnsheep had the highest number of CLs, whereas Texel had an average of 1.7 216 CLs (Supplementary Table S1). F1 showed phenotypes closer to those of Finnsheep than those of 217 Texel, having 3.75 CLs on average (Supplementary Table S1). We did not observe more than 2 CLs 218 in the Texel group or fewer than 3 CLs in Finnsheep or F1 cross-bred. Similarly, on average, 219 Finnsheep had the highest number of embryos (n=2.6), followed by F1 crosses (n=1.8) and Texel 220 (n=1.5). The F1 crosses displayed phenotypes similar to those of Finnsheep; this was unsurprising, as 221 we observed a similar pattern in an earlier study (Pokharel et al., 2018). Interestingly, the embryo 222 survival rate in Texel where 1.5 embryos were present from 1.7 CLs (88%) on average. On the other 223 hand, Finnsheep (63%) and F1 cross (48%) had remarkably low embryo survival rate. While these 224 findings are based on fewer animals, the results are in line with earlier studies (Rhind et al., 1980; 225 Silva et al., 2016) where typically higher litter size is associated with higher embryo mortality and 226 vice versa. It would be of great interest to determine if productivity follows the same pattern in F2 227 (i.e., F1 x F1) crosses, backcrosses and presumably also in a reciprocal cross.

228 4.2 RNA-Seq data 229 From the 42 libraries (21 from each tissue, including three technical replicates), 4.4 billion raw reads 230 were sequenced, of which 4.2 billion clean reads were retained after trimming. The summary 231 statistics from Trim Galore revealed that up to 3.6% of the reads were trimmed, with reverse-strand 232 reads having a comparatively higher percentage of trimmed bases. However, the percentage of reads 233 that were excluded for being shorter than 18 bp was always less than 1% across all samples 234 (Supplementary table S2). Up to 70% of the high-quality reads were mapped to the ovine reference 235 transcriptome (Ensembl release 92).

236 4.3 Gene expression in CL and endometrium 237 A total of 21,287 gene transcripts were expressed in the whole data set, of which 1,019 and 959 were 238 specific to the endometrium and CL, respectively. Genes such as cytochrome P450, family 11, 239 subfamily A, polypeptide 1 (CYP11A1), C-C motif chemokines (CCL21, CCL26), serpin family A 240 members (SERPINA1, SERPINA5), inhibin subunit alpha (INHA) and paternally expressed 10 241 (PEG10) were specific to CL while genes related with solute carriers (SLC44A4, SLC7A9, 242 SLC34A2), ERVs were endometrium-specific (Table 1). Further grouping of the expressed genes 243 showed that the most genes (n =19,440) were expressed in endometrium samples of Texel, while the 244 fewest genes (n =19,305) were expressed in endometrium samples of F1 crosses indicating s high 245 variation within the tissue. The cumulative difference in the number of genes in different samples and 246 tissues might be due to transcriptional noise. Nevertheless, the total number of genes expressed in 247 these tissues is comparatively higher than that in ovaries (Pokharel et al., 2018). As shown in Fig. 1, 248 the highest number of breed-specific genes expressed in the CL was found in Finnsheep (n=254), 249 followed by F1 crosses (n=204) and Texel (n=199). Similarly, from endometrium samples, we 250 observed the highest number of unique genes in F1 crosses (n=284), followed by Finnsheep (n=244) 251 and Texel (n=201). In a pairwise comparison, based on overall gene expression, Finnsheep and Texel 252 shared a higher number of genes (n=260) in the CL than in the endometrium. Moreover, Finnsheep 253 and F1 crosses were found to share relatively more common genes (n=278) than the other pairs (Fig. 1). 254 1).

255 Several Igs were expressed in the endometrium samples. Igs are heterodimeric proteins that belong to 256 the Ig superfamily (IgSF) (Williams and Barclay, 1988). Igs are composed of two heavy and two

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257 light chains, and the light chain may further consist of a κ or λ chain (Williams and Barclay, 1988). 258 Interestingly, the structure and organization of the genes enable Igs to be receptive to a virtually 259 unlimited array of antigens rather than being limited to a fixed set of ligands (Honjo, 1983). This 260 feature is particularly important for adaptation to changing environments and may have contributed 261 to enabling Finnsheep, for example, to survive in the harsh Finnish climate. Studies on humans have 262 shown that Igs, in general, improve pregnancy success (De Placido et al., 1994; Coulam and 263 Goodman, 2000). In addition to 11 Ig genes representing both the light and heavy chains, the joining 264 chain of multimeric IgA and IgM (JCHAIN) was also expressed. JCHAIN is a small polypeptide 265 containing eight cysteine residues that makes disulfide (C-C) bonds with IgA and IgM to form 266 multimers. Two of the eight cysteines are linked with cysteines available on the heavy chain of IgA 267 or IgM to result in dimer or pentamer forms, respectively (Bastian et al., 1995). We also identified 268 several genes associated with endogenous retroviruses (ERVs) in the endometrium samples. ERVs 269 are copies of retroviral genomes that have been integrated into the host genome during evolution. 270 Sheep ERVs share sequence similarity with exogenous and pathogenic Jaagsiekte sheep retrovirus 271 (JSRV) (DeMartini et al., 2003). The genome of sheep contains at least 32 ERVs related to JSRV 272 (Sistiaga-Poveda and Jugo, 2014), and these ERVs are essential during pregnancy, including during 273 placental morphogenesis and conceptus elongation (Palmarini et al., 2001; Dunlap et al., 2006b; 274 Spencer and Palmarini, 2012). A number of earlier studies have suggested critical roles of enJSRVs in 275 uterine protection from viral infection, preimplantation conceptus development and placental 276 morphogenesis (Dunlap et al., 2005, 2006b, 2006a; Denner, 2016). Interestingly, one of the novel 277 genes (ENSOARG00000009959) predicted to be an ERV was part of reduced FecL locus which is 278 linked to prolificacy in French Lacaune breed (Drouilhet et al., 2013). This gene is located on the 279 reverse strand of chromosome X and has 24 paralogs. This gene is not listed for 162 (out of 184) 280 species available in the Ensembl database. Although Ensembl lists 71 orthologs of this gene, none of 281 them have even 50% sequence homology. A BLAST search against the NR database showed that 282 97% of the bases matched to the region of the reduced FecL locus (GenBank ID KC352617.1), which 283 was recently characterized (Drouilhet et al., 2013). So far, only two genes, beta-1,4 N- 284 acetylgalactosaminyltransferase 2 (B4GALNT2) and insulin-like growth factor 2 mRNA-binding 285 protein 1 (IGF2BP1), and a pseudogene, ezrin-like protein, have been identified to exist in that 286 region; our results have added one more gene. In addition to the finding that the best hit was related 287 to the FecL locus, the gene appeared to be an ERV, as we noticed that the query gene had 98% 288 sequence identity with a partial sequence of the endogenous-virus beta-2 pro/pol region (see also Fig. 289 4). Finally, several lincRNAs were also expressed in the dataset. LincRNAs are long ncRNAs 290 (lncRNAs) that originate from intergenic regions and do not overlap a protein-coding transcript. 291 LincRNAs have a wide array of functions, including transcriptional regulation, biogenesis, epigenetic 292 regulation, tissue specificity and developmental patterning (see reviews by (Pauli et al., 2011; Ulitsky 293 and Bartel, 2013; Deniz and Erman, 2017; Ransohoff et al., 2018).

294 Although we observed considerable overlap of genes between tissues, principal component analysis 295 (PCA) of the 500 most highly expressed genes clearly indicated two distinct groups (Fig. 2A). 296 Similarly, a heatmap plot based on the top 25 genes with the highest levels of gene expression 297 variation across all samples showed a similar pattern (Fig. 2B). However, we did not observe any 298 breed-specific clusters in either of the tissues, which was also the case in our earlier ovarian 299 transcriptome study(Pokharel et al., 2018). In addition to distinctiveness in terms of gene expression, 300 the PCA plot also revealed that the CL samples appeared to be more homogeneous than the 301 endometrium samples. The two sub-clusters within the endometrium cluster is linked to the age of 302 the embryo (i.e. days after mating) and indicated the experimental bias. More importantly, sampling 303 bias owing to difference in days of collecting endometrium biopsies was apparent in the gene 304 expression. In general, samples in the upper right were older (13-16 days) compared to those in the

305 lower right. Such difference in gene expression has been attributed to the effects of interferon-tau 306 which causes massive changes in the endometrial gene expression starting day 13 of pregnancy (Gray 307 et al., 2006; Spencer et al., 2007; Forde and Lonergan, 2017). Therefore, we did not proceed further 308 with the differential gene expression comparisons of endometrium samples. However, similar bias 309 was not observed in CL.

310 Despite sharing 15 of the top 25 highly expressed genes between the tissues there were 5393 311 differentially expressed genes between endometrium and CL (Supplementary table S3). We noticed 312 that several genes belong to particular gene or protein family were upregulated exclusively in two 313 tissues. Cilia and flagella associated proteins (CFAP100, CFAP300, CFAP45, CFAP65), 314 desmosomes including two desmocollins (DSC1, DSC2), four desmogleins (DSG1, DSG2, DSG3) 315 and desmoplakin (DSP), were upregulated in endometrium. Members of homeobox A and B were 316 upregulated in endometrium while those from C and D were upregulated in CL. Thrombomodulin, 317 thrombospondins (THBS1, THBS2, THBS3, THBS4), thrombospondin type 1 domain containing 318 (THSD1, THSD7A, THSD7B), thromboxane A synthase 1 (TBXAS1) and thromboxane A2 receptor 319 (TBXA2R) were all upregulated in CL. Transforming growth factors (TGFB1, TGFB2, TGFB3), 320 TGFB receptors (TGFBR1, TGFBR2, TGFBR3), TGFB1 induced transcript 1 (TGFB1|1) and TGFB 321 induced (TGFBI) were upregulated in CL. Four genes associated with PDZ and LIM domain 322 (PDLIM2, PDLIM3, PDLIM4) and PDZ and LIM domain protein 2-like were upregulated in CL. 323 Guanylate binding proteins (n=7) were all upregulated in CL. EHD protein family comprises four 324 members and three (EHD2, EHD3, EHD4) were exclusively upregulated in CL. Three transcripts 325 (ENSOARG00000017142, ENSOARG00000014427, ENSOARG00000019903) belonging to 326 endogenous retrovirus group K were significantly upregulated in endometrium. Similarly, Placenta- 327 expressed transcript 1 protein and placenta-specific gene 8 protein-like were upregulated in 328 endometrium. Plakophilin 2 (PKP2) and 3 (PKP3), and plakophilin-1-like were also upregulated in 329 endometrium.

330 4.4 Most highly expressed genes 331 One of the most interesting findings was the significant interplay between the CL and endometrium 332 during the preimplantation phase, as revealed by the most highly expressed genes. To obtain an 333 overview of the most abundant genes in each tissue, we selected the top 25 genes (Table 2). We 334 noticed that fifteen out of the top 25 genes were shared in both tissues, and the majority (9 out of 15) 335 were mitochondrial genes. Mitochondrial genes play prominent roles during reproduction. We have 336 also observed high levels of expression of mitochondrial genes in ovaries during the follicular growth 337 phase (Pokharel et al., 2018). Six shared autosomal genes also appeared to play substantial roles 338 during the preimplantation stage. Translationally controlled tumor protein (TCTP) is a highly 339 conserved, multifunctional protein that plays essential roles in development and other biological 340 processes in different species (Tuynder et al., 2002; Chen et al., 2007; Brioudes et al., 2010; Li et al., 341 2011; Branco and Masle, 2019). With a maximum level of expression on Day 5 of pregnancy, this 342 protein has been shown to play a significant role in embryo implantation in mice (Li et al., 2011). 343 Consistent with these earlier studies, TCTP appeared to have the highest level of expression during 344 the embryo implantation period. Matrix Gla protein (MGP) is a vitamin K-dependent extracellular 345 matrix protein whose expression has been shown to be correlated with development and maturation 346 processes (Zhao and Nishimoto, 1996; Zhao and Warburton, 1997) and receptor-mediated adhesion 347 to the extracellular matrix (Loeser and Wallin, 1992). Several studies have reported that MGP is 348 highly expressed in the bovine endometrium (Spencer et al., 1999; Mamo et al., 2012; Forde et al., 349 2013). The high level of expression of MGP in our study is consistent with the results of earlier 350 studies in which this gene was found to be elevated during the preimplantation stage in sheep

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351 (Spencer et al., 1999; Gray et al., 2006) and cattle (Mamo et al., 2012). Similarly, (Casey et al., 2005) 352 reported that MGP was significantly upregulated in nonregressed compared to regressed bovine CLs. 353 Our data and supporting results from earlier studies on cattle show that MGP is highly expressed in 354 both tissues during the preimplantation stage and plays important roles in superficial implantation 355 and placentation in sheep. In summary, gene expression patterns in the CL and endometrium are 356 similar. 357 358 Six genes (NUPR1, BCL2L15, CST3, CST6, S100G, and OST4; see Table 2 for descriptions) specific 359 to the endometrium and one gene (B2M) common to both the CL and endometrium were also found 360 to be highly abundant in a recent study in which the authors compared gene expression changes in 361 the luteal epithelium and glandular epithelium during the peri-implantation stage in sheep (Brooks et 362 al., 2016). Galectin 15 (LGALS15) is induced by IFNT and is involved in conceptus development and 363 implantation (Kim et al., 2003; Gray et al., 2004; Lewis et al., 2007). LGALS15 mRNA has been 364 detected in ewes from Day 9 until Day 12 (Satterfield et al., 2006). IFNT is secreted by the ovine 365 conceptus trophectoderm during the middle to late luteal phase and acts as the signal for maternal 366 recognition of pregnancy. Furthermore, LGALS15 is an important gene that facilitates adhesion of the 367 trophectoderm to the endometrial luminal epithelium (Lewis et al., 2007; Spencer et al., 2007). Two 368 cystatin (CST) family members, namely, cystatin C (CST3) and cystatin E/M (CST6), were highly 369 expressed in the endometrium. Known for their importance during the elongation and implantation of 370 the conceptus, CSTs are protease inhibitors that are initiated by progesterone, and their high 371 expression levels are attributable to stimulation by IFNT (Spencer et al., 2008, 2015). Elongation 372 factor 1-alpha (EEF1A1) is an important component of the protein synthesis machinery because it 373 transports aminoacyl tRNA to the A sites of ribosomes in a GTP-dependent manner (Tatsuka et al., 374 1992; Mateyak and Kinzy, 2010). The high levels of expression of EEF1A1 in the endometrium most 375 likely correspond to the production and transport of progesterone and other molecules that are 376 essential during the implantation stage. The exact function of BCL2-like 15 (BCL2L15) in the sheep 377 endometrium is not known, nor has it been reported in the endometria of other species, but its high 378 expression has been reported previously (Koch et al., 2010; Brooks et al., 2016; Romero et al., 2017). 379 Oxytocin (OXT) was one of the most highly expressed genes in the CL. In cyclic ewes, OXT secreted 380 from the CL and posterior pituitary is widely known to bind with oxytocin receptor (OTR) from the 381 endometrium to concomitantly release prostaglandin F2α (PGF) pulses and induce luteolysis (Flint 382 and Sheldrick, 2004; Spencer et al., 2004a, 2004b; Bazer, 2013). However, for noncyclic ewes, OXT 383 plays an important role during peri-implantation and throughout pregnancy (Kendrick, 2000). OXT 384 signaling is known to be influenced by progesterone, but the mechanism underlying the regulation is 385 not yet clear due to conflicting findings (Grazzini et al., 1998; Gimpl et al., 2002; Fleming et al., 386 2006; Bishop, 2013). OTR expression in both the CL and endometrium was almost negligible 387 compared to OXT expression. Steroidogenic acute regulatory protein (STAR) plays an important role 388 in mediating the transfer of cholesterol to sites of steroid production (Stocco, 2000; Christenson and 389 Devoto, 2003). Post ovulation, the expression of the majority of genes associated with progesterone 390 synthesis starts to increase and peaks around the late luteal phase, when the CL has fully matured 391 (Juengel et al., 1995; Devoto et al., 2001; Davis and LaVoie, 2018). STAR, together with the 392 cytochrome P450 side chain cleavage (P450cc) complex and 3b-hydroxysteroid 393 dehydrogenase/delta5 delta4-isomerase (HSD3B1), are the three most important actors involved in 394 progesterone biosynthesis. STAR is involved in transporting free cholesterol to the inner 395 mitochondrial membrane. The P450cc complex, composed of a cholesterol side chain cleavage 396 enzyme (CYP11A1), ferredoxin reductase (FDXR) and ferredoxin (FDX1), converts the newly arrived 397 cholesterol into pregnenolone (King and LaVoie, 2009). Finally, HSD3B1 helps in converting 398 pregnenolone to progesterone (Hu et al., 2010; Plant et al., 2015; Stouffer and Hennebold, 2015;

399 Davis and LaVoie, 2018). Two of these major genes involved in progesterone synthesis (STAR and 400 HSD3B1) were ranked among the top 25 most highly expressed autosomal genes, while CYP11A1 401 (TPM=2,080), FDXR (TPM=86) and FDX1 (TPM=1,206) were also highly expressed.

402 4.5 Breed wise gene expression differences in the CL 403 Because of the sampling bias owing to the Overall, the CL appeared to display higher levels of gene 404 expression differences between the breeds than the endometrium (Table 3). This tissue-specific 405 difference is consistent with the findings of a recent study in cattle in which (Moore et al., 2016) 406 identified nine and 560 DEGs in the endometrium and CL, respectively, between fertile and infertile 407 cows. In the three possible pairwise comparisons for each tissue, the highest number of DEGs 408 (n=199) was found between the pure breeds in the CL, while the fewest DEGs (n=2) were found 409 between the Finnsheep and F1 crosses in the endometrium. In both tissues, the pure breed 410 comparisons had the highest numbers of DEGs, but the two comparisons (for both the endometrium 411 and CL) that involved the F1 crosses had the lowest. In other words, in the CL, Finnsheep had more 412 DEGs (n=67) than F1 crosses, whereas in the endometrium, Texel had more DEGs (n=17) than F1 413 crosses.

414 We compared pair-wise (Finnsheep vs Texel, Finnsheep vs F1 and Texel vs F1) differential gene 415 expression in the CL between three breeds. Out of the 199 significant DEGs in the CL of pure breeds 416 (i.e Finnsheep vs Texel), 140 were upregulated in Finnsheep (Supplementary table S4) and the rest 417 were downregulated. However, 91 out of the 199 genes lacked annotations (i.e., a gene name and 418 gene description). We were able to retrieve the CDSs for 82 genes and employed a BLAST search 419 against the NR database. Based on the Ensembl ncRNA prediction system, two out of nine genes that 420 lacked CDSs were predicted to be miRNAs, and the rest were lincRNAs.

421 In the list of DEGs, we observed a few cases in which more than one gene from the same family was 422 present. All eight genes related to multidrug resistance-associated proteins (MRPs) were upregulated 423 in Texel ewes, of which seven genes were type 4, while one was type 1. Both MRP1 and MRP4 are 424 lipophilic anion transporters. Earlier reports have suggested a role of MRP4 in transporting 425 prostaglandins in the endometrium (Lacroix-Pépin et al., 2011), and MRP4 has been found to be 426 upregulated in the endometrium in infertile cows compared to fertile cows (Moore et al., 2016). 427 Although there are no reports regarding the existence and roles of both MRP4 and MRP1 in the CL, 428 we speculate that the comparatively lower levels of these prostaglandin (PG) transporters in 429 Finnsheep provide a luteoprotective effect. Six sialic acid-binding Ig-like lectins (Siglecs) were 430 upregulated in Finnsheep. Based on a BLAST search and on the information available in Ensembl, 431 the sequences were related to SIGLEC-5 (ENSOARG00000002701), SIGLEC-13 432 (ENSOARG00000014846 and ENSOARG00000014850) and SIGLEC-14 (ENSOARG00000014875, 433 ENSOARG00000002909, and ENSOARG00000001575). Siglecs are transmembrane molecules that 434 are expressed on immune cells and mediate inhibitory signaling (Varki and Angata, 2006). So far, 435 SIGLEC-13 has been reported only in nonhuman primates; it was deleted during the course of human 436 evolution (Angata et al., 2004). The importance of Siglecs in immune system regulation has been 437 reviewed elsewhere (Pillai et al., 2012). Siglecs constantly evolve through gene duplication events 438 and may vary between species and even within a species (Cao and Crocker, 2011; Pillai et al., 2012; 439 Bornhöfft et al., 2018). Here we have reported the expression Siglecs in CL which are known to play 440 a role in the immune response during early pregnancy (preimplantation).

441 Similarly, three genes related to phospholipase A2 inhibitor and Ly6/PLAUR domain-containing 442 protein-like (PINLYP) were upregulated in Finnsheep. Other genes with more than one member

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443 included major histocompatibility complexes (MHCs) (BOLA-DQA5, HLA-DMA, HLA-DRA, MICA, 444 BOLA-DQB*2001, etc.), chemokines (CCL5, CXCL13, and CXCL9), solute carriers (SLC13A5, 445 SLC44A5, and SLC15A2), interleukin receptors (IL2RG, IL12RB1, and IL12RB2), cluster of 446 differentiation factors (CDs) (CD52, CD74, and CD300H), granzymes (GZMM and 447 LOC114109030/GZMH), calcium homeostasis modulators (CALHM3 and CALHM5) and 448 neurofilaments (NEFL and NEFM). Five out of seven significantly differentially expressed lincRNAs 449 were upregulated in Finnsheep. GZMM and GZMH have been found to be upregulated in Yakutian 450 cattle compared to Finncattle and Holstein cattle (Pokharel et al., 2019).

451 Out of 140 genes that were upregulated in Finnsheep, 50 genes (35.71%) were associated with 48 452 different GO terms (Fig. 3, Supplementary table S5) within the biological processes category, 453 whereas 90 genes lacked GO annotations. The upregulated genes were associated with positive 454 regulation of several processes such as “T cell migration”, “cytokine production”, “defense 455 response”, “immune system process”, “interferon-gamma production”, “leukocyte chemotaxis”, 456 “response to external stimulus”, , “cell–cell adhesion” and “cytokine-mediated signaling pathway”. 457 Some biological processes potentially associated with implantation were “maintenance of location”, 458 “plasma membrane invagination”, “import into cell”, “chemotaxis”, and “receptor internalization”. 459 Other biological processes, such as “response to bacterium”, “response to lipopolysaccharide”, 460 “lymphocyte-mediated immunity” and “chemotaxis”, could be associated with adaptation of 461 Finnsheep to the rugged Finnish climate and with disease resistance. In summary, genes involved in 462 the immune response were upregulated in Finnsheep CL during early pregnancy.

463 Similarly, only 40 of the 140 genes upregulated in Finnsheep were associated with 29 KEGG 464 pathways (Supplementary fig. 1, Supplementary table S6). The majority of the pathways were 465 associated with diseases; “tryptophan metabolism”, “cell adhesion molecules (CAMs)”, “Th1 and 466 Th2 cell differentiation” and “Th17 cell differentiation” appeared to play roles in implantation. Out 467 of 59 genes that were downregulated in Finnsheep, 17 and 14 genes were associated with GO IDs 468 and KEGG pathways, respectively. However, after applying our selection criteria (GO terms with 469 minimum level of 3 and maximum level of 5 whereby minimum of 3 genes and 3 % of genes from 470 given GO terms, also see methods section), only one biological process, “negative regulation of 471 endopeptidase activity” (associated genes: COL28A1, LOC101104482, and SLPI) and one KEGG 472 pathway, “bile secretion” (associated genes: LOC101106409, LOC101107772, and LOC101112460), 473 were identified. 474 Altogether, 67 genes were differentially expressed between Finnsheep and F1 crossbred ewes, of 475 which 49 genes were upregulated in Finnsheep (Supplementary table S7). CA5A is a member of the 476 carbonic anhydrase family of zinc-containing metalloenzymes, whose primary function is to catalyze 477 the reversible conversion of carbon dioxide to bicarbonate. The mitochondrial enzyme CA5A plays 478 an important role in supplying bicarbonate (HCO3-) to numerous other mitochondrial enzymes. In a 479 previous study, we observed downregulation of CA5A in the ovaries of Texel compared to F1 480 (Pokharel et al., 2018). More recently, CA5A was also shown to be expressed in the ovaries of the 481 Pelibuey breed of sheep; the gene was upregulated in a subset of ewes that gave birth to two lambs 482 compared to uniparous animals (Hernández-Montiel et al., 2019). However, there are no reports 483 regarding the expression and function of CA5A in the CL. Based on the results from our current and 484 earlier reports (Pokharel et al., 2018; Hernández-Montiel et al., 2019), CA5A appears to have an 485 important function, at least until the preimplantation stage of reproduction. The level of expression in 486 F1 crosses in the CL and endometrium followed the same pattern as that in the ovary, which led us to 487 conclude that CA5A is heritable and potentially an imprinted gene. Further experiments are needed to 488 determine whether the gene is associated with high prolificacy. Out of the 49 upregulated genes, 24

489 genes had available functional annotations and were associated with nine different GO terms (Fig. 5, 490 Supplementary table S8). The majority of the GO terms were related to transport (“anion transport”, 491 “lipid transport”, “organic anion transport”, and “fatty acid transport”) and regulation (“regulation of 492 lipid transport”, “regulation of homotypic cell–cell adhesion”, “negative regulation of T cell 493 activation”, and “regulation of lipid localization”).

494 Altogether, 20 out of the 49 genes upregulated in Finnsheep vs F1 crosses were linked to KEGG 495 pathways. Based on the selection criteria, only two KEGG pathways, namely, “complement and 496 coagulation cascades” (associated genes C5AR1, F13A1, and VSIG4) and “Fc gamma R-mediated 497 phagocytosis” (associated genes: FCGR1A, SCIN, and SYK), were identified. The lowest number 498 (n=22) of DEGs was observed between Texel and F1 crossbred ewes, with 13 genes being 499 upregulated in Texel (Supplementary table S9). 500 We noticed that several genes were differentially expressed in more than one comparison, increasing 501 our confidence in the identification of these DEGs. Few DEGs were exclusively up- or 502 downregulated in particular breed compared to other two breeds. For example, a transcript encoding 503 a miRNA (ENSOARG00000022916) was found to be always upregulated in Finnsheep compared to 504 both Texel and F1 crosses. HNRNPK was always downregulated in Finnsheep, and CA5A was always 505 upregulated in F1 crosses compared to the other two breeds. Similarly, coiled-coil domain-containing 506 73 (CCDC73) and a pseudogene (ENSOARG00000020196) were upregulated in Texel compared to 507 F1 crosses. A lincRNA (ENSOARG00000025875) was downregulated in Finnsheep compared to 508 Texel. MICA* was upregulated in Finnsheep compared to Texel. Oxidized low-density lipoprotein 509 receptor 1 (OLR1), NLR family apoptosis inhibitory protein (NAIP), macrophage scavenger receptor 510 1 (MSR1), high-affinity Ig gamma Fc receptor 1 precursor (FCGR1A), hemoglobin subunit alpha-1/2, 511 folate receptor 3 (FLOR3*), Fc gamma 2 receptor, chromogranin B (CHGB), Siglec-14, clavsin 2 512 (CLVS2), copine 4 (CPNE4), EPH receptor B6 (EPBH6*) and MICA* were exclusively upregulated 513 in the CLs of Finnsheep. Gastrula zinc finger XICGF17.1-like (LOC10562107), crystallin mu 514 (CRYM), myeloid-associated differentiation marker-like (LOC101119079) and tolloid-like 2 (TLL2) 515 were downregulated in the CLs of Texel compared to the other two breeds. These results also 516 indicated that F1 crosses were more similar to Finnsheep than Texel crosses. CA5A appeared to be 517 upregulated in F1 crosses compared to both Finnsheep and Texel from both phases (i.e., in the CL in 518 this study and in the ovary in our earlier study).

519 4.6 miRNAs expressed in the dataset 520 A total of 336.6 M reads were sequenced, of which approximately 42% contained adapters and/or 521 low-quality bases. After trimming, more than 92% of the reads (n=311.3 M) were retained as high- 522 quality clean reads. On average, collapsing of duplicate reads revealed 483,096 unique reads per 523 sample, of which 54.4% of the unique sequences (collapsed reads) were mapped to the ovine 524 reference genome. The detailed summary statistics for each sample are shown in Supplementary table 525 S10. There were more collapsed reads and uniquely mapped reads for endometrial samples than CL 526 samples despite the similar numbers of raw and clean reads in both tissues. After filtering out low- 527 count (<10 reads) and ambiguous reads, a total of 599 miRNAs were included in the expression 528 analysis. All the miRNAs quantified in this study are presented in Supplementary table S11 and have 529 been sent to be considered for adding in the next release of miRBase. The majority of the expressed 530 miRNAs (n=524) were shared by both tissues, with 43 and 32 miRNAs being unique to the CL and 531 endometrium, respectively. Out of 599 miRNAs, 60 were conserved miRNAs in other species while 532 123 were known sheep miRNAs. Currently, 153 miRNAs are available in the miRBase database 533 (Kozomara et al., 2019). The database was updated to the current version (22) from an earlier version 534 (miRBase 21) after four years, and the overall number of miRNA sequences increased by over a

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535 third. However, the number of sheep miRNAs remained the same. Moreover, studies that produce 536 miRNA datasets have been scarce. As of April 2019, miRNA datasets from only three studies were 537 available in the European Nucleotide Archive (ENA) database, with accession codes PRJNA308631 538 (n=3), PRJEB22101 (n=37) and PRJNA414087 (n=40); the PRJEB22101 dataset was from the first 539 phase of this study (Pokharel et al., 2018). In the current study, we quantified over threefold more 540 sheep miRNAs (n=599) than are available in miRBase. Therefore, these miRNAs will certainly 541 improve the existing resources and will be valuable in future studies. 542 We did not perform differential gene expression analysis on the endometrial samples because of the 543 sampling bias. Two miRNAs, both upregulated in Finnsheep, were significantly differentially 544 expressed between the pure breeds in the CL, while the other comparisons did not reveal any 545 significantly differentially expressed miRNAs. Of these two significantly differentially expressed 546 miRNAs, rno-miR-451-5p is a conserved miRNA similar to one found in rats (Rattus norvegicus). 547 The other, oar-18_757_mt, is a novel miRNA expressed on chromosome 18. Chromosomal 548 placement of the quantified miRNAs revealed a large cluster of miRNAs on chromosome 9 that we 549 also observed in the ovaries (Fig.5).

550 4.7 Limitations and thoughts for future studies 551 We acknowledge certain limitations of this study. We believe that with the availability of a better 552 annotated reference genome, the data from this study will reveal additional information that we may 553 have missed in this analysis. With sequencing costs becoming increasingly inexpensive, increasing 554 the sample size of each breed group would certainly add statistical power. Given that time-series 555 experiments are not feasible with the same animal, sampling could be performed with a larger group 556 of animals at different stages of pregnancy to obtain an overview of gene expression changes. One 557 ovary from each ewe was removed earlier (Pokharel et al., 2018) and all the CLs for this study was 558 collected from the remaining ovary. Therefore, there might be some impact due to possible negative 559 feedback effects on overall gene expression. It should be noted that overall gene expression and, 560 more specifically, differential expression between breeds is inherently a stochastic process; thus, 561 there is always some level of bias caused by individual variation (Hansen et al., 2011). After noticing 562 the bias caused by pregnancy length on endometrium, we were unable to proceed further with breed- 563 wise differential expression analyses. Including more individuals in future experiments as well as 564 more controlled tissue sampling will minimize such bias. We are also aware that the overall gene 565 expression profiles may have been affected by the absence of one CL that was removed for earlier 566 study, (Pokharel et al., 2018). Having said that, as CL was removed from all ewes in current study, 567 we do not expect any bias in the gene expression comparison. The results from breeding experiments 568 have shown that productivity traits such as litter sizes may not carry on to F2 crosses (F1 x F1) 569 and/or backcrosses. Therefore, future experiments that involve F2 crosses and backcrosses would 570 provide more valuable findings related to prolificacy. Moreover, by doing a reciprocal cross 571 experiment, we might be able to get insight into POE and measure the potential contribution of the 572 Texel and Finnsheep in each cross. In addition, replicating such experiments in different 573 environments would be relevant for breeding strategies to mitigate the effects of climate change. To 574 minimize or alleviate noise from tissue heterogeneity, single-cell experiments may prove beneficial 575 in future studies. While we observed interplay between the endometrium and the CL, it would be 576 equally interesting to measure the transcriptional patterns in the embryo. Finally, the application of 577 gene-modifying technologies such as CRISPR/Cas9 to edit certain regions (such as the region in the 578 FecL locus homologous to the partial retrovirus sequence) may provide important insights into 579 phenotypes associated with infertility, prolificacy and other traits of interest.

580 5 Conclusion

581 We compiled the most comprehensive list thus far of genes (n=21,287) and miRNAs (n=599) 582 expressed in the CL and endometrium, which are the most important tissues during the 583 preimplantation stage and therefore determine the success of pregnancy in sheep. Our results agree 584 well with the (limited) existing reports, which are mainly focused on the interplay of the 585 endometrium and conceptus, but we have shown that the CL plays an equally important role. The 586 relative scarcity of transcriptomic information about the CL means that its functional importance is 587 underrated. We identified several key transcripts, including coding genes (producing mRNA) and 588 noncoding genes (miRNAs, snoRNAs, and lincRNAs), that are essential during early pregnancy. 589 Functional analysis primarily based on literature searches and earlier studies revealed the significant 590 roles of the most highly expressed genes in pregnancy recognition, implantation and placentation. F1 591 crosses were more closely related to Finnsheep than to Texel, as indicated by phenotypic and gene 592 expression results that need to be validated with additional experiments (with F2 crosses and 593 backcrosses). Several genes with potential importance during early pregnancy (including SIGLEC13, 594 SIGLEC14, SIGLEC6, MRP4, and CA5A) were reported in the CL for the first time in any species. 595 The roles of retroviruses during early pregnancy and in breed-specific phenotypes were indicated by 596 the observed gene expression dynamics, especially in the endometrium. A novel gene sharing 597 similarity with an ERV was identified in the FecL locus. The results from this study show the 598 importance of the immune system during early pregnancy. We also highlight the need for improved 599 annotation of the sheep genome and emphasize that our data will certainly contribute to such 600 improvement. We observed a cluster of miRNAs on chromosome 18 homologous to that found on 601 chromosome 14 in humans. Taken together, our data provide new information to aid in understanding 602 the complex reproductive events during the preimplantation period in sheep and may also have 603 implications for other ruminants (such as goats and cattle) and mammals, including humans.

604 6 Abbreviations

605 Ig (immunoglobulin), Siglec (sialic acid-binding Ig-like lectin), ERV (endogenous retrovirus), CDS 606 (coding sequence), OXT (oxytocin), MRP (multidrug resistance-associated protein), CL (corpus 607 luteum), lincRNA (long intergenic noncoding RNA), TPM (Transcripts Per Million)

608 7 Conflict of Interest

609 The authors declare that the research was conducted in the absence of any commercial or financial 610 relationships that could be construed as potential conflicts of interest.

611 8 Author Contributions

612 J.K. and M.H.L. conceived and designed the project. J.K., M.H. and J.P. collected the samples. K.P. 613 analyzed the data and wrote the manuscript. J.P. contributed substantially to revising the manuscript. 614 M.W. and J.K. contributed to the data analysis and manuscript writing, respectively. All authors 615 revised and approved the final manuscript.

616 8.1 Funding 617 This study was funded by the Academy of Finland (decisions 250633, 250677 and 285017). This 618 study is part of the ClimGen (“Climate Genomics for Farm Animal Adaptation”) project funded by 619 FACCE-JPI ERA-NET Plus on Climate Smart Agriculture. K.P. acknowledges financial support 620 from the Niemi Foundation.

621 9 Acknowledgments

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622 We are grateful to Anu Tuomola for providing the experimental facilities on her farm in Urjala and to 623 Kati Kaisaioki from Lallin Lammas Ltd. for providing the slaughtering facilities. We thank Johanna 624 Rautiainen, Arja Seppälä, Annukka Numminen, Kalle Saastamoinen, Ilma Tapio, Tuula Marjatta 625 Hamama, Anneli Virta and Magnus Andersson for their valuable assistance during this study. The 626 authors wish to acknowledge the CSC – IT Center for Science, Finland, for computational resources. 627 This study was supported by the Finnish Functional Genomics Centre of the University of Turku, 628 Åbo Akademi and Biocenter Finland.

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955 Xu, S.-S., Gao, L., Xie, X.-L., Ren, Y.-L., Shen, Z.-Q., Wang, F., et al. (2018). Genome-Wide 956 Association Analyses Highlight the Potential for Different Genetic Mechanisms for Litter 957 Size Among Sheep Breeds. Frontiers in Genetics 9, 118. doi:10.3389/fgene.2018.00118.

958 Yang, J., Li, X., Cao, Y.-H., Pokharel, K., Hu, X.-J., Chen, Z.-H., et al. (2018). Comparative mRNA 959 and miRNA expression in European mouflon (Ovis musimon) and sheep (Ovis aries) 960 provides novel insights into the genetic mechanisms for female reproductive success. 961 Heredity. doi:10.1038/s41437-018-0090-1.

962 Zhao, J., and Nishimoto, S. K. (1996). Matrix Gla protein gene expression is elevated during 963 postnatal development. Matrix biology: journal of the International Society for Matrix 964 Biology 15, 131–40.

965 Zhao, J., and Warburton, D. (1997). Matrix Gla protein gene expression is induced by transforming 966 growth factor-beta in embryonic lung culture. American Journal of Physiology-Lung Cellular 967 and Molecular Physiology 273, L282–L287. doi:10.1152/ajplung.1997.273.1.L282.

968

969 11 FIGURE LEGENDS

970 Figure 1 Venn diagram showing the distribution of genes expressed in the A) CL and B) 971 endometrium of Finnsheep, Texel and F1 crosses.

972 Figure 2 Sample relatedness. (A) PCA plot of the top 500 expressed genes in the CL (left) and 973 endometrium (right) and (B) heatmap of the top 25 most variable genes across all samples. Tissue- 974 specific samples are denoted with a trailing c (for CL) or e (for endometrium). Legend: FS – 975 Finnsheep; TX – Texel ewes, F1 – F1 crosses of Finnsheep and Texel sheep

976 Figure 3 GO terms associated with the list of genes that were upregulated in the CLs of Finnsheep 977 compared to Texel

978 Figure 4 Multiple sequence alignment (partial) of the novel endogenous retrovirus gene. (A) The 979 novel ERV identified in this study belongs to FecL locus and is located between B4GALNT2 and 980 Enzrin-like protein. (B) The multiple sequence alignment was prepared using Clustal Omega 981 (Madeira et al., 2019) based on novel ERV (nERV, ENSOARG00000009959), ovine endogenous- 982 virus beta-2 pro/pol region, partial sequence (kERV, AY193894.1), Ovis canadensis canadensis 983 isolate 43U chromosome 17 sequence (OC43U, CP011902.1) and the reverse complement of reduced 984 FecL locus (RFecL, KC352617). The bases are colored based on the nucleotide coloring scheme in 985 Jalview (Waterhouse et al., 2009). 986 Figure 5 miRNA clusters in sheep (Chr. 18, top) and humans (Chr. 14, bottom). Only three (marked 987 in black font color) out of 46 miRNAs in this cluster were not expressed in our data.

988 12 TABLES

989 Table 1: Top 25 genes (ranked by TPM) exclusively expressed in CL and endometrium.

TPM Gene name Chr. Gene description CL ENSOARG00000003867 2080.2 CYP11A1 18 cytochrome P450, family 11, subfamily A, polypeptid

24 This is a provisional file, not the final typeset article bioRxiv preprint doi: https://doi.org/10.1101/673558; this version posted November 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International Sheeplicense. preimplantation transcriptome

ENSOARG00000009107 1233.5 CCL21 2 C-C motif chemokine ligand 21 ENSOARG00000013402 1004.3 PTGFR 1 prostaglandin F receptor ENSOARG00000013340 622.3 CCL26 24 C-C motif chemokine ligand 26 ENSOARG00000019663 424.4 PTHLH 3 parathyroid hormone like hormone ENSOARG00000015144 273.6 SERPINA5 18 serpin family A member 5 ENSOARG00000004774 248.0 LOC101114790* 11 C-C motif chemokine 15 ENSOARG00000014882 225.6 SERPINA1 18 serpin family A member 1 ENSOARG00000009230 201.1 DPT 12 dermatopontin ENSOARG00000016052 188.9 AOX1 2 aldehyde oxidase 1 ENSOARG00000008189 177.9 PKIB 8 cAMP-dependent protein kinase inhibitor beta ENSOARG00000004455 177.6 LHCGR 3 luteinizing hormone/choriogonadotropin receptor ENSOARG00000020188 160.0 FAM110B* 9 Ovis aries family with sequence similarity 110 memb ENSOARG00000009597 151.2 HS6ST2* X heparan sulfate 6-O-sulfotransferase 2 ENSOARG00000000474 143.3 GJA4 1 gap junction protein alpha 4 ENSOARG00000002475 131.9 PEG10 4 paternally expressed 10 ENSOARG00000010344 130.1 LTBP1 3 latent transforming growth factor beta binding protein ENSOARG00000020976 118.9 GLDN 7 gliomedin ENSOARG00000014966 116.3 INSL3 5 insulin like 3 ENSOARG00000009887 111.7 CHST15 22 carbohydrate sulfotransferase 15 ENSOARG00000017273 108.7 PROS1 1 protein S ENSOARG00000020243 104.9 INHA 2 Ovis aries inhibin subunit alpha (INHA), mRNA. ENSOARG00000019971 99.6 PLN 8 phospholamban ENSOARG00000005118 92.8 KCNK12 3 potassium two pore domain channel subfamily K mem ENSOARG00000016448 90.1 TFR2 24 transferrin receptor 2 Endometrium ENSOARG00000004013 2378.2 FXYD4 25 FXYD domain containing ion transport regulator 4 ENSOARG00000012900 1429.8 IGFBP1 4 insulin like growth factor binding protein 1 ENSOARG00000002255 1191.2 19 acyl-coenzyme A thioesterase THEM4-like ENSOARG00000019845 1078.1 3 enJSRV-20 endogenous virus Jaagsiekte sheep retrov ENSOARG00000015448 654.7 LOC114115259* 1 endogenous retrovirus group K member 6 Pro protein ENSOARG00000014187 589.7 HamM* 19 endogenous virus Jaagsiekte sheep retrovirus ENSOARG00000011311 544.3 CLDN7 11 claudin 7 ENSOARG00000010828 436.5 HAVCR1* 5 hepatitis A virus cellular receptor 1 ENSOARG00000004021 420.7 GRP* 23 gastrin releasing peptide ENSOARG00000003992 389.0 SLC44A4 20 solute carrier family 44 member 4 ENSOARG00000019903 367.3 14 endogenous retrovirus group K member 9 Pol protein ENSOARG00000004279 333.7 SLC7A9 14 solute carrier family 7 member 9 ENSOARG00000018755 327.1 TNNI1 12 troponin I1, slow skeletal type ENSOARG00000013020 316.0 HSD11B1 12 hydroxysteroid 11-beta dehydrogenase 1 ENSOARG00000003224 300.3 SMPDL3B 2 sphingomyelin phosphodiesterase acid like 3B ENSOARG00000012115 266.9 15 Jaagsiekte sheep retrovirus-like element ENSOARG00000013158 263.8 SPDYC 21 speedy/RINGO cell cycle regulator family member C ENSOARG00000006119 257.5 MMP7 15 matrix metallopeptidase 7 ENSOARG00000004751 246.5 TMEM92 11 transmembrane protein 92 ENSOARG00000010191 234.5 PEBP4 2 phosphatidylethanolamine binding protein 4 ENSOARG00000007680 232.5 SLC34A2 6 solute carrier family 34 member 2 ENSOARG00000004297 216.7 ALOX12* 11 arachidonate 12-lipoxygenase, 12S type

ENSOARG00000007592 211.8 PTGS2 12 prostaglandin-endoperoxide synthase 2 ENSOARG00000008009 211.1 CDH17 9 cadherin 17 ENSOARG00000014451 204.7 IFI27L2 18 interferon alpha-inducible protein 27-like protein 2 990

991 Table 2: List of the 25 most abundant genes in the CL and endometrium. Fifteen of the top 25 992 genes were shared by both tissues and were dominated by mitochondrial genes. The table includes 993 the Ensembl gene ID, chromosome number (Chr.), gene ID (GeneID) and gene description. The table 994 is divided into three sections; the first section lists the 25 genes that were shared by the two tissues, 995 and the other two list the remaining 10 genes in the endometrium and CL. Gene IDs and annotations 996 that were not available in BioMart were retrieved based on a homology search using the nucleotide 997 BLAST (marked with an asterisk, “*”) or on information available in Ensembl (marked with a hash, 998 “#”).

Chr. GeneID Description Common ENSOARG00000007815 6 LOC105580399* Cercocebus atys 60S ribosomal protein L41-like ENSOARG00000000019 MT COX2 cytochrome c oxidase subunit II ENSOARG00000018666 7 RPLP1 ribosomal protein lateral stalk subunit P1 ENSOARG00000000035 MT CYTB cytochrome b ENSOARG00000000021 MT ATP8 ATP synthase F0 subunit 8 ENSOARG00000007617 10 TCTP tumor protein, translationally controlled 1 ENSOARG00000000023 MT COX3 cytochrome c oxidase subunit III ENSOARG00000003793 23 TSMB4X thymosin beta-4 ENSOARG00000000037 MT Mt tRNA# mitochondrial tRNA ENSOARG00000020724 3 MGP matrix Gla protein ENSOARG00000000016 MT COX1 cytochrome c oxidase subunit I ENSOARG00000000022 MT ATP6 ATP synthase F0 subunit 6 ENSOARG00000000028 MT ND4 NADH dehydrogenase subunit 4 ENSOARG00000003782 7 B2M beta-2-microglobulin ENSOARG00000000006 MT ND1 NADH dehydrogenase subunit 1

Endometrium ENSOARG00000019088 AMGL01125506.1 LGALS15 lectin, galactoside-binding, soluble, 15 ENSOARG00000003184 24 NUPR1 nuclear protein 1, transcriptional regulator ENSOARG00000019924 1 BCL2L15 BCL2-like 15 ENSOARG00000006202 13 CST3 cystatin C ENSOARG00000021079 1 S100A11 S100 calcium-binding protein A11 ENSOARG00000016080 13 ATP5F1E PRELI domain-containing 3B ENSOARG00000013018 X S100G S100 calcium-binding protein G ENSOARG00000019491 3 OST4 oligosaccharyltransferase complex subunit 4, noncatalytic ENSOARG00000001346 21 CST6 cystatin E/M ENSOARG00000006149 8 EEF1A1 eukaryotic translation elongation factor 1 alpha 1

CL ENSOARG00000004595 13 OXT oxytocin/neurophysin I prepropeptide ENSOARG00000022293 13 RF02216# misc. RNA ENSOARG00000000027 MT ND4L NADH dehydrogenase subunit 4L ENSOARG00000002586 15 APOA1 apolipoprotein A1

26 This is a provisional file, not the final typeset article bioRxiv preprint doi: https://doi.org/10.1101/673558; this version posted November 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International Sheeplicense. preimplantation transcriptome

ENSOARG00000002472 25 MSMB microseminoprotein beta ENSOARG00000001269 26 STAR steroidogenic acute regulatory protein ENSOARG00000000010 MT ND2 NADH dehydrogenase subunit 2 ENSOARG00000013157 X TIMP1 TIMP metallopeptidase inhibitor 1 ENSOARG00000020402 1 HSD3B1 hydroxy-delta-5-steroid dehydrogenase, 3 beta- an steroid delta-isomerase 1 ENSOARG00000000033 MT ND6 NADH dehydrogenase subunit 6 999

1000 Table 3: Numerical summary of differentially expressed genes in the CL and endometrium. Legend: 1001 FS – Finnsheep, TX – Texel, F1 – F1-cross

Comparison CL Endometrium

Upregulated Downregulated Upregulated Downregulated

FS vs TX 140 59 22 21

FS vs F1 49 18 2 0

TX vs F1 13 9 5 12

1002

1003 13 Supplementary Material

1004 Fig. S1: KEGG pathways associated with the DEGs upregulated in Finnsheep compared to Texel in 1005 the CL

1006 Fig. S2: GO terms associated with DEGs upregulated in Finnsheep compared to F1 crosses

1007 Fig. S3: PCA of the top 500 expressed miRNAs in the CL (left) and endometrium (right)

1008 Table S1: Phenotype data of the samples

1009 Table S2: Summary of the samples included in mRNA-Seq

1010 Table S3: List of DEGs between the CLs of Finnsheep and Texel ewes

1011 Table S4: List of DEGs between the CL and endometrium

1012 Table S5: List of GO terms associated with the upregulated genes in the CLs of Finnsheep compared 1013 to Texel

1014 Table S6: List of KEGG pathways associated with the upregulated genes in the CLs of Finnsheep 1015 compared to Texel

1016 Table S7: List of DEGs between the CLs of Finnsheep and those of F1 crosses

1017 Table S8: List of GO terms associated with the upregulated genes in the CLs of Finnsheep compared 1018 to F1 crosses

1019 Table S9: List of DEGs between the CLs of Texel and F1 crosses

1020 Table S10: Summary of the miRNA-Seq data

1021 Table S11: List of miRNAs quantified in this study

1022 Data Availability Statement

1023 The raw FASTQ sequence data (for both mRNAs and miRNAs) from this study have been deposited 1024 in the European Nucleotide Archive (ENA) database under accession code PRJEB32852. The 1025 accession codes for each sample are included in the sample summary tables (Supplementary table S3 1026 and S10 for mRNA and miRNA, respectively).

28 This is a provisional file, not the final typeset article bioRxiv preprint doi: https://doi.org/10.1101/673558; this version posted November 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

208 278 254 204 244 284 18700 18672 260 215 226 206

199 201

A B Finnsheep Texel F1 crosses F1 FS TX F1 FS TX breed 5 group − 5 0 . The copyright holder for this preprint (which was The copyright holder for

ENSOARG00000020402 ENSOARG00000002586 ENSOARG00000009107 ENSOARG00000014882 ENSOARG00000015144 ENSOARG00000001269 ENSOARG00000013402 ENSOARG00000004455 ENSOARG00000010688 ENSOARG00000002472 ENSOARG00000003867 ENSOARG00000004595 ENSOARG00000013018 ENSOARG00000004279 ENSOARG00000020433 ENSOARG00000019924 ENSOARG00000008468 ENSOARG00000007680 ENSOARG00000019845 ENSOARG00000004013 ENSOARG00000014100 ENSOARG00000003992 ENSOARG00000002255 ENSOARG00000019088 ENSOARG00000012900 974e

50 4519e 897e 4823e 48e 4271e 379e 554e 302e 4590e 312e 73e 251e 0 107e

CC-BY-NC-ND 4.0 International license CC-BY-NC-ND 4.0 International 3609e this version posted November 14, 2019. this version posted November

; 1033e PC1: 87% variance 4563e under a 4208e 897c 4563c 4519c 554c 4590c 3609c 4271c −50 974c 4823c https://doi.org/10.1101/673558 48c 1033c doi: 4208c 379c 312c 302c 73c

0 251c not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available bioRxiv a license to display the preprint in perpetuity. is the author/funder, who has granted not certified by peer review)

20 40

bioRxiv preprint

−40 −20 variance PC2: 7% 7% PC2: 107c

A B bioRxiv preprint doi: https://doi.org/10.1101/673558; this version posted November 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

%Genes / Term 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 positive regulation of cytokine production 8** regulation of cytokine-mediated signaling pathway 3* adaptive immune response 7** myeloid leukocyte activation 3* lymphocyte activation involved in immune response 5** lymphocyte-mediated immunity 5* antigen processing and presentation of exogenous peptide antigen 3** positive regulation of immune system process 12** positive regulation of leukocyte chemotaxis 4** regulation of leukocyte activation 9** positive regulation of immune effector process 3* positive regulation of adaptive immune response 3* chemotaxis 8** amine catabolic process 3** response to bacterium 9** cytokine-mediated signaling pathway 7** calcium-mediated signaling 3. second messenger-mediated signaling 5* cyclic nucleotide-mediated signaling 3* regulation of cell-cell adhesion 8** cellular cation homeostasis 6* T cell differentiation 5* positive regulation of defense response 5* receptor internalization 3* positive regulation of response to external stimulus 9** response to lipopolysaccharide 4* positive regulation of interferon-gamma production 3* homotypic cell-cell adhesion 12** T cell activation 12** indole-containing compound catabolic process 3** defense response to bacterium 4* leukocyte activation 13** negative regulation of GPCR signaling pathway 3** positive regulation of smooth muscle cell proliferation 3* regulation of lymphocyte proliferation 7** regulation of behavior 5** regulation of T cell activation 7** release of sequestered calcium ion into cytosol 4** maintenance of location 5* leukocyte aggregation 12** cellular response to interferon-gamma 4** granulocyte chemotaxis 3* myeloid leukocyte migration 4* cell-cell adhesion 14** import into cell 8** plasma membrane invagination 4** alpha-amino acid catabolic process 4** positive regulation of T cell migration 3** bioRxiv preprint doi: https://doi.org/10.1101/673558; this version posted November 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Novel ERV (120555 - 122258) 1 10 K 20 K 30 K 40 K 50 K 60 K 70 K 80 K 90 K 100 K 110 K 120 K 130 K 140 K 150 K 160 K 170 K 180 K 194,639

Genes B4GALNT2 (19173 - 81032) IGF2BP1 (146903 - 186435) AGH33861.1 mRNA-beta-1,4 N-acetylgal... AGH33862.1 insulin-like growth factor...

A Enzrin-like protein (133689 - 136376)

OC43U ------T C A C C A T Y T CG T S GG A A A A C T T G T A G C C A C T G C T C A G A T T C C T Y G A G C T C T C C CWT T K A A A T GG T T A A C T G A T G T Y C C T A A A T GG R T nERV A A G T A C A GG A A A A A G A A C A A T G C T C A C T A T C T CG T CGG A A A A C T T G T A G C C A C T G C T C A A A T T C C T CG A G C T C T C C C A T T G A A A T GG T T A A C T G A T G T C C C T A A A GGGG T RFecL ------G A A C A A C A C T C A C C A T G T CG T CGG A A A A C T T G T A G C C A C T G C T C A G A T T C C T CG A G C T C T C C C A T T G A A A T GG T T A A C T G A T G T C C C T A A A T GGG T kERV ------G A A C A A C A C T C A C C A T C T CG T CGG A A A A C T T G T A G C C A C T G C T C A G A T T C C T CG A G C T C T C C C A T T G A A A T GG T T A A C T G A T G T C C C T A A A T GGG T

OC43U T G A G C A R T K G CWG C T T C C A A A A G T G A A G C T CG A GG C T T T A G A A C A A T T A G T A A A A G A A C A G C T T C A A T C T GG C C A T R T T R A A Y C T T C T A C R T C T C C T T GG A A T T C T C C T G nERV T G A G C A G T GG C CG C T T T C A A A A G T G A A G C T CG A GG C T T T A A A A C A A T T A G T A A A A G A A C A G C T T C A A T C T GG C C A T A T T G A A C C T T C T A CG T C T C C T T GG A A T T C T C C T G RFecL T G A G C A G T GG C CG C T T C C A A A A G T G A A G C T CG A GG C T T T A G A A C A A T T A G T A A A A G A A C A G C T T C A A T C T GG C C A T A T T A A A C C T T C T A CG T C T C C T T GG A A T T C T C C T G kERV T G A G C A G T GG C CG C T T C C A A A A G T G A A G C T CG A GG C T T T A G A A C A A T T A G T A A A A G A A C A G C T T C A A T C T GG C C A T A T T G A A C C T T C T A CG T C T C C T T GG A A T T C T C C T G

OC43U T T T T T G T C A T T A A G A A A A A A T C T GG T A A A T GG A G A A T G T T RWC T G A T T T A A GGGM A G T T A A C A A A T G T A T A R A A C C T A T GGG A G C T T T G C A A T T A GG T C T C C C T T C T C C T nERV T T T T T G T C A T T A A G A A A A A A T C T GG T A A A T GG A G A A T G T T A A C T G A T T T A A GGG C A G T T A A C A A A T G T A T A G A A C C T A T GGG A G C T T T G C A A T T A GG T C T C C C T T C T C C T RFecL T T T T T G T C A T T A A G A A A A A A T C T GG T A A A T GG A G A A T G T T G A C T G A T T T A A GGG C A G T T A A C A A A T G T A T A G A A C C T A T GGG A G C T T T G C A A T T A GG T C T C C C T T C T C C T kERV T T T T T G T C A T T A A G A A A A A A T C T GG T A A A T GG A G A A T G T T G A C T G A T T T A A GGG C A G T T A A C A A A T G T A T A G A A C C T A T GGG A G C T T T G C A A T T A GG T C T C C C T T C T C C T

OC43U G C T T T G A T T C C A C A GG A T T GG T C T T T G A T RG T T T T A G A T T N N N N N N A T T G T T T T T T T T T T T T T T T A T A T T C C T T T G C A A A T A A A A G A T A G A A A T A A A T T T G C T K T T A C T A nERV G C T T T G A T T C C A C A GG A T T GG T C T T T G A T GG T T T T A G A T T T G A A GG A T T G T T ------T T T T - - - T A T T C C T T T G C A A A T A A A A G A T A G A A A T A A A T T T G C T T T T A C T A RFecL G C T T T G A T T C C A C A A G A T T GG T C T T T G A T GG T T T T A G A T T T G A A GG A T T G C T ------T T T T T A A T A T T C C T T T G C A A A T A A A A G A T A G A A A T A A A T T T G C T T T T A C T A kERV G C T T T G A T T C C A C A GG A T T GG T C T T T G A T GG T T T T A G A T T T G A A GG A T T G T T ------T T T T T A A T A T T C C T T T G C A A A T A A A A G A T A G A A A T A A A T T T G C T T T T A C T A

OC43U T T C C A G T G T A T A A T C A T GGG C A G C C CG T A A A A Y G T T A T C A A T GG A C A G T G T T G C C T C A A GG A A T G A T T A A T A G C C C T A C A C T T T G T C A GG A G T T T G T T R A T Y G T G C T C T T nERV T T C C A G T G T A T A A T C A T GGG C A G C C CG T A A A A CG T T A T C A A T GG A C A G T G T T G C C T C A A GG A A T G A T T A A T A G C C C T A C A C T T T G T C A GG A G T T T G T T A A T CG T G C T C T T RFecL T T C C A G T G T A T A A T C A T GGG C A G C C CG T A A A A CG T T A T C A A T GG A C A G T G T T G C C T C A A GG A A T G A T T A A T A G C C C T A C A C T T T G T C A GG A G T T T G T T G A T CG T G C T C T T kERV T T C C A G T G T A T A A T C A T GGG C A G C C CG T A A A A CG T T A T C A A T GG A C A G T G T T G C C T C A A GG A A T G A T T A A T A G C C C T A C A C T T T G T C A GG A G T T T G T T A A T CG T G C T C T T

OC43U A T T A C T G T G A G A C A A C A A T T T T C T A A T T G T C T T C T C T A T C A T T A T A T A G A T G A T C T T C T A T T GG C A G C T C C T A G T A A A G A R R A A C R A G A T A M A T T T T T T T A T T C A T G T R A nERV A T T A C T G T G A G A C A A C A A T T T T C T A A T T G T C T T C T C T A T C A T T A T A T A G A T G A T C T T C T A T T GG C A G C T C C T A G T A A A G A GG A A CG A G A T A C A T T - T T T T A T T C A T G T A A B RFecL A T T A C T G T G A G A C A A C A A T T T T C T A A T T G T C T T C T C T A T C A T T A T A T GG A T G A T C T T C T A T T GG C A G C T C C T A G T A A A G A GG A A C A A G A T A A A T T - T T T T A T T C A T G T G A kERV A T T A C T G T G A G A C A A C A A T T T T C T A A T T G T C T T C T C T A T C A T ------bioRxiv preprint doi: https://doi.org/10.1101/673558; this version posted November 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

64.61Mb 64.62Mb 64.63Mb 64.64Mb 64.65Mb 64.66Mb 64.67Mb Chr. 18 oar-mir-379 oar-mir-494 oar-mir-376e oar-mir-539 oar-mir-382 oar-mir-496 Forward strand oar-mir-411a oar-mir-1193 oar-mir-376c oar-mir-544 oar-mir-134 oar-mir-377 oar-mir-380 oar-mir-543 oar-mir-376d oar-mir-655 oar-mir-668 oar-mir-541 Genes (Ensembl) oar-mir-411b oar-mir-495 oar-mir-376a oar-mir-3959 oar-mir-154a oar-mir-323c

oar-mir-1197 oar-mir-3958 oar-mir-3956 oar-mir-487a oar-mir-154b oar-mir-656 oar-mir-323a oar-mir-654 oar-mir-485 oar-mir-3957 oar-mir-758 oar-mir-376b oar-mir-323b oar-mir-409 oar-mir-329b oar-mir-1185 oar-mir-412 oar-mir-329a oar-mir-381 oar-mir-369 oar-mir-487b oar-mir-410 Contigs H.sap-O.ari lastz-net AMGL01043260.1

Chr. 18 Reverse strand Chr. 14 101.01Mb 101.02Mb 101.03Mb 101.04Mb 101.05Mb 101.06Mb 101.07Mb H.sap-O.ariChr. lastz-net.14 Forward strand H.sap-O.ari lastz-net MIR376C MIR1185-2 MIR655 MIR382 MIR154 MEG9 MIR379 MIR758 MIR1193 MIR376A2 MIR381HG MIR487A MIR496 MIR541 MIR411 MIR329-2 MIR543 MIR654 MIR381 MIR134 MIR377 MIR410

Genes MIR299 MIR494 MIR495 MIR376B MIR487B MIR668 MIR409 (Genecode 29) MIR380 MIR376A1 MIR539 MIR485 MIR412 MIR1197 MIR300 MIR889 MIR323B MIR369 MIR323A MIR1185-1 MIR544A MIR656 MIR329-1 Contigs AL132709.5 Chr. 14 Reverse strand